Isolation of sterols



Patented Oct. 30, 1951 ISOLATION or s'rEnoLs Willy Lange, Cincinnati,and Robert G. mu..-

logen, Golf Manor, Ohio, assignors to The Procter & Gamble Company,Cincinnati, Ohio,

'acorporaflon oi. Ohio No Drawing. Application June 25, 1948,

Serial No. 35,283

This invention relates to the treatment of sterol-containing materials,and more particularly to the recovery of typical sterols containing the3(beta)-hydroxy-5z6-ene grouping from materials such as wool fat, fishliver oils, soybean oil, cottonseed oil, tallow, other vegetable andanimal oils and fats, gallstones, nerve tissue, and othersterol-containing substances.

An object of the present invention is to provide a process for therecovery of typical sterols containing the 3(beta) -hydroxy-5:6-enegrouping from sterol-containing substances.

A further object is to provide a process for the separation of sterolscontaining the 3(beta)- hydroxy-5z6-ene bond grouping from derivativesthereof, such as oxidation and reduction prod-p uct's, which do notcontain both the 3(beta) -hydroxy group and the 5:6 double bond.

Another object is to provide a rapid and continuous process for theprecipitation of sterols of particular concern herein from solutions ofsame in oxygen-free solvents.

Methods for the recovery of sterols have been proposed heretofore, butas far as we are aware there is no method which permits theprecipitation of sterols in an inexpensive and rapid operationespecially adapted for use on a commercial scale. For example,digitonin, which is used in sterol laboratories to precipitate sterols,is very expensive and is therefore used only for the separation of smallquantities of materials. Since digitonln precipitates not only thesterols themselves, but also many types of sterol derivatives,

subsequent fractionation and separation of the recovered precipitatedmaterial may be necessary if pure sterols are desired.

A method for the isolation of cholesterol has been proposed by Natelsohnet al. in U. S. Patent 2,220,114. The method is based on the formationof true esters of cholesterol with polybasic acids such as sulfuric,phosphoric and boric acids,

11 Claims. (Cl. 260-3973) anhydrous hydrogen chloride or bromide attemperatures between 5 and 20 C. The process requires considerable timeand exposes the sterol to the action of strong acid for extended periodswith ensuing dehydroxylation and consequent reduction in sterol yield.

. In another patent, U. S. 2,362,605, Yoder describes the precipitationof sterols from an oxygen-free solvent with anhydrous oxalic acid. The

method requires considerable time and istedious.

In accordance with the present invention, typical sterols such ascholesterol, phytosterol, stigmasterol, beta-sitosterol, ergosterol andalso 'l-dehydrocholesterol, all of which possess the 3-(beta)-hydroxy-5:6-ene grouping, are precipitated rapidly and completelywith perchloric acid,

H0104, or hexafluorophosphoric acid, HPFc, from suitable solutions aswell-formed, white crystals, which are believed to be sterol-acidaddition products and from which the sterol may be suborhexafluorophosphoric acid of a concentration followed by the formationof alkali metal salts involves the separation of sterols from theunsaponifiable fraction recovered from wool grease. The unsaponifiablematerial is dissolved in a nonalcoholic solvent and sterols areprecipitated with Impurities are then refrom about per cent to about percent, preferably in limited excess, to precipitate the true sterolsubstantially completely. The precipitated crystals-oi the sterol-acidaddition product, which .iorm almost instantaneously and from which; thesterol may be recovered with unchanged structure, are thereafterpromptly separated 'byifilltration, centriiugation, or otherwise from;theymother liquor containing soluble sterol derivatives in solution. Thecrystalline residue thereafter treated with water or an aqueous alkalinesolution, preferably in the presence of a water-insoluble solvent forthe sterol. This treatment efiects hydrolysis of the sterol-acidaddition product, the liberated sterols being dissolved by the solvent.The resulting solvent solution is preferably washed with water until thewash water is neutral, and the sterols are recovered by evaporation ofthe solvent.

If recovery of the soluble sterol derivatives is desired, the motherliquor resulting from the above separation is also washed with water oraqueous alkaline solutions or both until neutral.

amazes it is preferable to effect washing or neutralization promptlyafter separation from the crystalline product. It is customary, forexample, to discharge the filtrate directly into water or an aqueousalkaline solution. The liberated sterol derivatives, in solventsolution, are separated from the aqueous phase and upon evaporation ofthe solvent, sterol derivatives are concentrated in the residue.

Sterols which may be recovered in accordance with the present inventionhave the basic ring structure of the cyclopentano-perhydrophenanthrenesmodified as follows:

a on,

2 l 8 nok 5 6 7 where R is cm H(CH:)r-CH(CH:):

or substitution products thereof.

These sterols, it will be noted, are characterized by the presence of a3(beta)-hydroxy group and a 5:6-ene bond. They may contain an additionaldouble bond in the ring system, for example in the 7:8 position. Ourwork has indicated that when the 3(beta) -hydroxy group and the 5:6double bond are both free and unblocked in the sterol molecules, thenthe acid addition products thereof, as formed in our invention, areinsoluble in the oxygen-free solvent, and separation of the sterol fromthose derivatives in which either the hydroxy group or the double bondhas been modified can be effected by the process of this invention- Itis to be borne in mind that hydrogenation of sterol-containingsubstances may effect conversion of the sterols to dihydrosterols inwhich the 5:6 double bonds do not exist and which do not precipitatefrom solution on reaction with the acid. 0n the other hand, ergosteroland its closely related derivative, 7-dehydrocholesterol, whichcompounds possess not only the above essential 3(beta) -hydroxy groupand 5:6-ene bond but also an additional double bond in the 7 :8position, are precipitated as white acid addition products by the acidsherein used. The presence of such second double bonds in the basic ringsystem, however, while not affecting the ability of the compound to formcrystallized precipitates with the acid, renders the compound especiallysensitive toward excess acid and care must be taken in acid treatment ofsuch materials to avoid substantial excesses of acid and hightemperature of treatment whereby the sterol may be decomposed.

3(beta) -hydroxy group and the 5:6 double bond, thereby permitting readyand rapid separation of typical sterols from said derivatives, anoperation of commercial value in the manufacture of hormones andvitamins. The acid treatment and separation of crystallized acidaddition product may be performed in a few minutes and completeseparation and recovery of the sterols involving hydrolysis of thecrystallized material may be performed in a relatively short time, not

longer than a few hours, as compared with the heretofore customarylengthy period involving elaborate work. Such permissible shorter timesof contact with excess concentrated acid are especially advantageous inthe event recovery of the sterol derivatives from the mother liquor isdesired because such derivatives have been found to decompose withrapidity in the presence of excess acid. However, it is to be noted thatadvantages are also realized with respect to the sterols themselves.These materials lose their hydroxy groups and thus lose their sterolcharacter in the presence of excess concentrated acid, albeit at asomewhat lower rate than their acidsensitive derivatives. Consequentlyany existing tendencies toward decomposition are held to a minimum withresulting increase in yield of valuable product.

Minimization of the tendency of sterols and sterol derivatives todecompose in the presence of excess precipitating acid is also aided byavoiding treatment temperatures substantially in excess of 100 F.Ordinary room temperatures from 70 to F. are of course preferred andlower temperatures eifected by refrigeration may be employed if desired,provided unsaponifiable materials remain in solution in the solvent atsuch lower temperatures.

In the recovery of sterol-like compounds wherein the 3-hydroxy group hasbeen esterifled, as in wool grease for example, preliminary hydrolysisof this ester group as by saponlflcation with sodium or potassiumhydroxide is necessary prior to treatment with the precipitating acid,otherwise the sterol is not precipitated. The liberated sterols andother unsaponiflable constituents of the saponifled mixture may beseparated from the soap by solvent extraction with the solvent inaccordance with known procedures. The solvent extract, preferably aftersuitable concentration, may then be subjected to treatment with the acidto precipitate the sterols.

It is to be observed also, for example, that the concentration ofsterols in naturally occurring substances, such as vegetable oils, isrelatively low, normally from 0.05 per cent to 0.1 per cent, and that atsuch low concentration and in the presence of oxygen-containingsubstances (glycerides or other esters) it is impractical to precipitatethe sterols by the direct addition of perchloric or hexafluorophosphoricacid even though the hydroxy group and double bond of the sterol may beunchanged. Accordingly in working with such sterol-containing substancesit is essential to extract the unsaponifiable material (which includesthe sterols) with a suitable solvent an then effect precipitation of thesterols.

Similarly, in the treatment of sterol-rich materials such as nervetissue in accordance with our invention, the sterols, along with otherunsaponifiable materials, are first extracted with the solvent toprovide a suitable solution from which the sterols may be subsequentlyprecipitated by the addition of the perchloric or hexafluorophosphoricacid.

d The solvents which may be employed as media for the precipitationreaction are those normally excess. of ergosterol which is verysensitive toward strong vents are petroleum ether, hexane, heptane,

chloroform, bromoform, carbon tetrachloride, chloroethylene,dichloroethylene, tri'chloroethylene, trichloropropane, 1,4dichlorobutane, benzene, toluene, monochlorobenzene, and the like.Saturated hydrocarbon solvents and their derivatives are particularlysuitable for use. It is especially desirable that the solvents be freefrom oxygen-containing solvents such as ethanol and acetone because suchmaterials are excellent solvents for the sterol-acid addition productsand tend to interfere with precipitation.

The amount of solvent relative to the sterol content of the mixture isnot critical, but it is preferable that the sterol concentration be nottoo low, that is, not less than about 2 percent. A concentration fromabout 5 per cent to about 20 derivatives when used in This is especiallynoticeable in the case the sterols and sterol acids.

Hexafiuorophosphoric acid, however, has one disadvantage in that it iscapable of forming a crystalline hydrate which may precipitate alongwith the sterol-acid addition product. If, for example, just enoughhexafluorophosphoric acid is removed from the acid phase of, the mixtureby combination with sterol to reduce the concentration of the acid tothe range of about 50-55 per cent, hydrate crystals of the acid form andprecipitate with the crystals of the sterol-acid addition product.Therefore, in the use of hexafiuorophosphoric acid it is preferable toadjust the amount and/or concentration of the acid so per cent sterol insolvent is preferred. Large'excesses of solvent of course reduce theconcentra-v tion of the sterols and tend to lower the percentage yieldobtained in precipitation.

The concentration of the acid employed is not critical. However,concentrations from 55 per cent to 85 per cent are preferably used. Ingeneral, more complete precipitation is effected by the use of higherconcentrations, but the destruction of the sterol and especially thesterol derivatives is more rapid under such conditions especially sinceexcesses are preferably used for maximum yield. It follows, therefore,that the importance of holding contact times to a minimum is greaterwith use of the more concentrated acids. We have found thatprecipitation ofthe sterols proceeds most satisfactorily when theconcentration of the acid employed is about 70 per cent. 1

The amount of acid (figured as anhydrous) desirable for use in effectingprecipitation is likewise not critical, but we have observed that theamount required for optimum. precipitation increases with decrease inthe concentration of the sterol in the' solvent. Although the productprecipitated is believed to be the result of the com-.

bination of one mol of acid with one mol of sterol, somewhat greaterthan the theoretical amount of acid is preferably employed in order torealize optimum yield even when concentrations of the sterol in thesolvent are high. If, for example, the concentration of the sterol inthe solvent is around 10 per cent, about 1 mols of acid is used toobtain substantially complete precipitation. A sterol concentration ofabout 5 per cent in the solvent, on the other hand, requires about 2mols of acid per mol of sterol. In some circumstances as much as 3 molsof acid per mol of sterol may be required, but larger amounts are notrecommended because of the increased tendency of the excess acid todestroy the sterol and especially the sterol derivatives.

Although either perchloric or hexafluorophosphoric acid servesadequately in the practice of the invention, hexafluorophosphoric acid,HPFs,

' has an advantage in that the dry sterol-acid addition products thereofare exceptionally stable and do not burn'when heated as do thecorresponding addition products formed from perchloric acid, HC1O4.Furthermore, hexafluorophosphoric acid is a weaker acid than perchloricacid and therefore has less destructive action on cipitation processwell adapted for continuous.

operation wherein continuously flowing, proportioned streams of thesterol-containing solvent solution and acid are intimately contacted ina suitable mechanical mixer to effect precipitation of the sterol-acidaddition product which is then separated from the mother liquor bycentrifugal separation. Neutralization of the sterol-containingprecipitate and the sterol-derivative-containing mother liquor withrecovery or concentration of :the separated constituents may then beeffected as described above.

The sterol-acid addition products are relatively stable in the absenceof excess concentrated acid and may be recovered as an excess-acid-free,dry

v appended claims.

Example 1.-A solution of 10 grams of cholesterol in 100 c. c. ofchloroform was vigorously agitated at 75-85 F. with about 5.44 grams of70% perchloric acid (about 1 mols HClO4 per mol of cholesterol).Crystallization was noted immediately. The mixture was stored at 75-80F. for about 1 minute, then filtered through an acid resistant filterwith the aid of suction. Care was taken to avoid sucking moist airthrough the cake of crystals for an appreciable length of theoreticalamount.

As an auxiliary example, hexane substituted for chloroform as thesolvent.

Example 2.-A solution of grams cholesterol in 100 c. c. chloroform wastreated 'as in Example ,lwith 4.32 grams of 70% perchloric acid (about2.3 moIs I-ICIOi per mol of cholesterol). The recoveredcrystals :weretreated with an aqueous sodium. hydroxide solution, the cholesterolbeing liberated in substantially theoretical amount.

- Example 3.-A solution of 10 grams phytosterols (a mixture of varioussterols) in 100 c. c. chloroform was treated as in Example 1 with 6.1grams of 70% perchloric acid (about 1.6 mols H0104 per mol ofphytosterol). The recovered crystals contained 23.21% HC104. Thephytosterols were liberatedon treatment of the crystals with an aqueoussodium hydroxide solution and separation thereof from the aqueous phasewas effected with the aid of chloroform as a solvent. Example4.-A'solution of approximately 1.0

gram of ergosterol in 35 c. c. of chloroform was thoroughly mixed at'75-80 F. with .39 gram of 70% perchloric acid per one gram ofergosterol. Instantaneously a white crystalline precipitate formed. Thiscrystalline material was separated by suction filtration and storedunder moisturefree conditions and 89.9% of theoretical yield wasobtained. The crystals contained 2o.20% HCIO4 whereas. the theoreticalacid content of ergosterol "H0104 is 20.22%. The ergosterol wasliberated from the crystalline material by treatment with an aqueoussodium hydroxide solution andthereafter extracted with chloroform.

: --E:campZe 5.-In the steam deodorization of fats .and fatty oils afat-like material is frequently .distilled off. This'material issometimes referred in the industry as clabber stock. Such ma- ;terialscontain sterol and sterol derivatives which may berecovered by meaiiaofthe present invention.- 100 grams of clabbei; stock were ,treated withcaustic soda solution\ to saponify -;the-. saponifiable materialcontained therein. ,Fromthe mixture produced, 20.1 grams ofunsaponifiable material were extracted with the use of hexane. Therecovered hexane solution was evaporated and the residue was blown withsteam undervacuum to remove volatile components. ..This,residue wascooled to Hf-80 F. and diluted with chloroform to 100 c. c. and 3.5grams of 70% perchloric acid were added. The mixture was vigorouslyagitatedeiCrystal- ,lization of the sterols was instantaneous and thecrystallizedgnaterial was recovered by suction filtration? 1? yield of3.2 grams of crystalline material was obtained corresponding to about"2.5% sterol in the original clabber stock.

Since the original unsaponifiablc material contained oxidation andhydrogenation products of sterols, these materials-did not precipitateon "treatment with perchloric acid but were conceritrated in the motherliquor. Upon neutralization of the mother liquor and evaporation of thesolvent, the sterol derivatives were recovered in the residuesubstantially free from 'sterols.

' Example 6.A solution of 1 gram cholesterol in 10 c. c. chloroform wasvigorously agitated at ordinary room-temperature with 0.58 gram of 65.2%hexafluorophosphoric acid (about 1 mol HPFc per mol" of cholesterol) Amass of white crystals formed immediately and was separated drumthe'motherliquor asin Example 1. The

for the mol-mol addition product.

recovered crystals analyzed as 26.90% HPFaal compared with a calculatedcontent of 27.43%

The ch01.- esterol liberated on treatment with lye solution wasrecovered by extraction with chloroform.

Having thus described our invention, what we claim and desire to secureby Letters Patent is:

1. A process for the separation of sterol containing a hydroxy group in3(beta) -position and a double bond in 5:6-position fromsterol-containing material, which comprises providing a solution of theunsaponifiable fractionof said material in an oxygen-free, liquidsolvent of the group consisting of hydrocarbons and halo.- genatedderivatives thereof. mixing therewith at a temperature not substantiallyin excess of 100 F. an acid of the group consisting of perchloric andhexafluorophosphoric acids thereby efiecting precipitation of sterolconstituents of the unsaponifiable fraction without effectingprecipitation of sterol derivatives, :and separating the precipitatefrom the mother liquor. 1 2. Process of claim 1 in which theconcentration of sterol in solvent is at least 2 per cent. 3. Process ofclaim 1 in which the acid concentration is not less than per cent andnot higher than 85 per cent.

4. Process of claim 3 in which the amount of acid ori an anhydrous basisdoes not substantially exceed 3 mols of acid per mol of sterol.

5. Process of claim 4 in which the acid is perchloric acid and in whichthe amount of an anhydrous basis does not substantially exceed 3 mols ofacid per mol of sterol.

6. Process of claim 4 in which the acid is hexafiuorophosphorlc acid andin which the amount on an anhydrous basis does not substantially exceed3 mols of acid per mol of sterol and gives a residualhexafluorophosphoric acid concentration below 50% in the mother liquorafter combination with sterol.

7. Process for the separation of sterol containing a 3-hydroxy group anda 5:6 double bond from sterol-containing material, which comprisesproviding a solution of the unsaponifiable..

v fraction of said material in an oxygen-free,

. per cent thereby effecting precipitation of sterol constituents of theunsaponiflable trac tion without effecting precipitation of sterolderivatives, separating the precipitate from. the mother liquor,hydrolyzing the precipitate and recovering the liberated sterol.

8. Process of claim '7 in which the concentration of sterol in solventis at least 2 per cent. i

9. Process of claim '7, in which thehydrolysis is effected by treatmentwith an aqueous alkaline solution. I

10. Proces of claim 9,"in which the treatment is effected in thepresence of a sterol solvent insoluble in water.

11. Process of separating sterol containinga 3-hydroxy group with a 5:6double bond from derivatives not containing both the 3-hydroxy group andthe 5:6-double bond, whichcomprises providing a solution of a mixture ofsterol and sterol derivative in an oxygen-free, liquid solvent of thegroup consisting of hydrocarbons and halogenated derivatives thereof,mixing with the solution at -a temperature not substantially in excessof 100 F. an acid of the group consisting of perchloric andhexafluoro'phosphoric acids in which the acid concentration is not lessthan 55 per cent and not higher than 85 per cent thereby effectingprecipitation of sterol without effecting precipitation of derivativesthereof, promptly separating the precipitate from the mother liquor,treating the precipitate with an aqueous alkaline solution therebyliberating sterol, recovering liberated sterol, neutralizing the motherliquor with an aqueous alkaline solution, sepa rating the aqueous phasefrom the solvent solution, and evaporating the solvent thereby obtaininga residue containing the sterol derivatives.

WILLY LANGE. ROBERT G. FOLZENLOGEN.

10 REFERENCES CITED The following references are of record in the fileof this patent:

UNITED s'rA'Ins PATENTS Number Name Date 2,220,114 Natelson Nov. 5, 19402,322,906 Yoder June 29, 1943 2,362,605 Yo'der Nov. 14, 1944 OTHERREFERENCES Hoffmann. Berichte 43, pp. 1080-1086 (1910).

1. A PROCESS FOR THE SEPARATION OF STEROL CONTAINING A HYDROXY GROUP IN3(BETA)-POSITION AND A DOUBLE BOND IN 5:6-POSITION FROMSTEROL-CONTAINING MATERIAL, WHICH COMPRISES PROVIDING A SOLUTION OF THEUNSAPONIFIABLE FRACTION OF SAID MATERIAL IN AN OXYGEN-FREE, LIQUIDSOLVENT OF THE GROUP CONSISTING OF HYDROCARBONS AND HALOGENATEDDERIVATIVES THEREOF, MIXING THEREWITH AT A TEMPERATURE NOT SUBSTANTIALLYIN EXCESS OF 100* F. AN ACID OF THE GROUP CONSISTING OF PERCHLORIC ANDHEXAFLUOROPHOSPHORIC ACIDS THEREBY EFFECTING PRECIPITATION OF STEROLCONSTITUENTS OF THE UNSAPONIFIABLE FRACTION WITHOUT EFFECTINGPRECIPITATION OF STEROL DERIVATIVES, AND SEPARATING THE PRECIPITATE FROMTHE MOTHER LIQUOR.